CN116769021B

CN116769021B - Monoclonal antibody for Vp7 protein of African horse sickness virus and application

Info

Publication number: CN116769021B
Application number: CN202310969810.7A
Authority: CN
Inventors: 独军政; 户鑫兵; 徐婧; 宋昱庆; 王轩莹; 田占成; 关贵全; 罗建勋; 殷宏
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2024-04-19
Anticipated expiration: 2043-08-03
Also published as: CN116769021A

Abstract

The invention belongs to the technical field of immunology and in-vitro diagnosis, and particularly relates to a monoclonal antibody aiming at Vth (African horse sickness virus) VP7 protein and a preparation method thereof. According to the invention, a recombinant expressed African horse sickness virus VP7 protein is immunized into a BALB/c mouse, a monoclonal antibody aiming at the African horse sickness virus VP7 protein is obtained through screening, and a nucleotide sequence for encoding heavy chain and light chain variable regions of the antibody is successfully obtained through a reverse transcription PCR technology. The monoclonal antibody has good affinity reaction capability with the VP7 protein expressed by recombination. The monoclonal antibody is used for establishing an African horse sickness virus competition ELISA antibody detection method, and provides a technical basis for effectively preventing and controlling the African horse sickness from being transmitted into China.

Description

Monoclonal antibody for Vp7 protein of African horse sickness virus and application

Technical Field

The invention belongs to the technical field of immunology and in-vitro diagnosis, and particularly relates to a monoclonal antibody aiming at an African horse sickness virus VP7 protein, a preparation method and application thereof.

Background

African horse sickness is a non-contact acute or subacute infectious disease of equine animals such as horses, donkeys, mules, etc. caused by African horse sickness virus (African horse sickness virus, AHSV). Horses are most susceptible to african horse sickness, young horses are most susceptible, donkey and mule are second, and the horses are generally asymptomatic after zebra infection and probably are storage hosts, and play an important role in the transmission process of the disease. The disease is transmitted mainly by biting midges. Clinical symptoms of african horse sickness can be classified into 4 types, pulmonary: latency is 3-5 days, and is typically characterized by respiratory symptoms, with foam-like secretions in the nostrils, which die within hours of clinical symptoms. Heart type: initial manifestation of fever, subsequent edema of the cheeks, orbit, neck and chest, and finally heart failure. Mixing: the mixed presence of lung and heart types is often found in post-mortem examinations. Heating type: clinical symptoms are not obvious and mainly exist in animals such as donkey, zebra and the like. The world animal health organization (WOAH) lists it as a disease that must be reported, and China lists it as a type of animal disease.

African horse sickness virus belongs to the genus Cyclovirus of the reoviridae family, whose genome consists of 10 double stranded RNA fragments of different sizes, encoding together 7 structural proteins (VP 1-VP 7) and 4 non-structural proteins (NS 1-NS 4), wherein VP2 is a serotype specific antigen and VP7 protein is a serogroup specific antigen. It has been found that there are at least 9 serotypes of African horse sickness virus and that VP7 protein-based ELISA methods can be used for detection of African horse sickness virus population-specific antibodies, suitable for large-scale sample detection and epidemiological investigation.

The current method for detecting African horse sickness antibodies mainly comprises competition ELISA, virus neutralization test, agar gel immune diffusion reaction and immunofluorescence test. The present invention screens and obtains a monoclonal antibody aiming at AHSV VP7 protein and variable region sequences of heavy chain and light chain thereof, and establishes an AHSV competitive ELISA antibody detection method by using the antibody as a competitive antibody.

Disclosure of Invention

Aiming at the technical problems, the invention provides a monoclonal antibody aiming at the Vth horse sickness virus VP7 protein, which has the characteristics of high titer, strong affinity and good specificity, and can specifically bind with the Vth horse sickness virus VP7 protein. The method specifically comprises the following steps:

In a first aspect, the present invention provides a monoclonal antibody directed against the VP7 protein of the african horse sickness virus, the monoclonal antibody comprising an antibody heavy chain and an antibody light chain;

the variable region CDR of the heavy chain of the monoclonal antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.5, a CDR2 with an amino acid sequence shown as SEQ ID NO.6 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 7;

The variable region CDR of the monoclonal antibody light chain comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 10.

Preferably, the amino acid sequence of the variable region of the heavy chain of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO. 3.

In a second aspect, the invention provides a nucleic acid encoding an antibody heavy chain and an antibody light chain of the monoclonal antibody of the first aspect above.

Preferably, the variable region of the heavy chain of the antibody has a gene sequence shown in SEQ ID NO.2 and the variable region of the light chain of the antibody has a gene sequence shown in SEQ ID NO. 4.

In a third aspect, the invention provides an application of the monoclonal antibody in the first aspect in preparing a reagent, a test strip or a kit for detecting african horse sickness virus.

In a fourth aspect, the present invention provides an immunoconjugate comprising:

(i) The monoclonal antibody of the first aspect;

(ii) And a coupling moiety selected from the group consisting of: a detectable label, a drug, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

In a fifth aspect, the invention provides an ELISA kit for African horse sickness virus, which is characterized in that the kit comprises the monoclonal antibody in the first aspect.

Preferably, the kit comprises an ELISA plate, african horse sickness virus antigen, a blocking solution, a diluent, the monoclonal antibody of claim 1 or 2, an ELISA secondary antibody, a washing solution, a color reagent and a stop solution.

Preferably, the african horse sickness virus antigen is selected from african horse sickness virus VP7 recombinant proteins.

Preferably, the blocking solution is a PBST buffer containing 5% nonfat dry milk;

preferably, the diluent is 0.01M PBS pH7.2;

Preferably, the wash solution is a PBST buffer.

Preferably, the enzyme-labeled secondary antibody is an HRP-labeled goat anti-mouse secondary antibody.

In a sixth aspect, the present invention provides the use of a monoclonal antibody according to the first aspect above for in vitro detection of african horse sickness virus for the purpose of non-disease diagnosis.

In a seventh aspect, the present invention provides a method for ELISA detection of African horse sickness virus for the purpose of diagnosis of a non-disease, said method comprising the steps of:

(1) Coating: diluting the expressed AHSV VP7 recombinant protein to 0.5 mug/mL by using a CBS buffer solution, coating 100 mug/hole into an ELISA plate, and coating at 4 ℃ overnight; washing the plate 5 times with PBST buffer; the CBS buffer was 0.05M carbonate-bicarbonate buffer, ph9.6;

(2) Closing: blocking the ELISA plate with PBST buffer containing 5% skimmed milk powder, 200 μl/well, and incubating at 37deg.C for 1 hr; washing the plate 5 times with PBST buffer;

(3) And (3) detection: african horse sickness positive serum, standard positive serum and standard negative serum are added into an ELISA plate, 100 μl/hole is incubated for 1h at 37 ℃, and the plate is washed 5 times with PBST buffer;

(4) Then adding 1:2000 dilution of the monoclonal antibody of the first aspect, incubating for 1h at 37 ℃, and washing the plate with PBST buffer for 5 times; the dilution was 0.01M PBS pH7.2;

(5) Adding enzyme-labeled secondary antibodies: 1, 1:40000 diluted HRP-labeled goat anti-mouse secondary antibody is added to an ELISA plate, 100 mu L/well, and incubated at 37 ℃ for 1h; washing the plate 5 times with PBST buffer; the dilution was 0.01M PBS pH7.2;

(6) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; 100 μl/well of stop solution was added and the OD ₄₅₀ was read;

The judging method comprises the following steps: blocking rate (BP) = (negative control OD ₄₅₀ -sample OD ₄₅₀)/(negative control OD ₄₅₀ -positive control OD ₄₅₀) ×100%; the positive control is less than 0.2, and the negative control is more than 2.0; when BP is less than or equal to 45%, the result is negative; when BP is more than or equal to 50%, the result is positive; when 45% < BP <50%, the results are suspicious.

The beneficial effects of the invention are as follows: ① The invention provides a monoclonal antibody for resisting VP7 protein of African horse sickness virus; ② Immunoblotting experiments show that the polypeptide reacts with structural protein VP7 specifically; ③ The monoclonal antibody can be specifically combined with the African horse sickness virus in infected cells, and can be used for detecting and diagnosing the African horse sickness virus; ④ Compared with the existing antibody detection method, the method has better sensitivity and specificity, and is suitable for detecting and diagnosing the African horse sickness virus.

Drawings

FIG. 1 identification of molecular weight and purity of AHSV VP 7-resistant monoclonal antibodies;

FIG. 2 immunofluorescence detection of AHSV VP 7-resistant monoclonal antibodies;

FIG. 3 immunoblot detection of anti-AHSV VP7 monoclonal antibodies.

Detailed Description

The following detailed description of embodiments of the invention is provided for the purpose of illustration only and is not to be construed as limiting the invention. In addition, all reagents employed in the examples below are commercially available or may be synthesized in accordance with text or known methods, as would be readily available to one skilled in the art for reaction conditions not listed, if not explicitly stated.

EXAMPLE 1 preparation of monoclonal antibodies against the VP7 protein of African horse sickness Virus

1. Immunization of mice

6 Female BALB/c mice of 6-8 weeks old were immunized with the expressed AHSV VP7 recombinant protein. The recombinant VP7 protein was emulsified with an equal amount of Freund's complete adjuvant, injected subcutaneously on the back, 50. Mu.g/dose. The first immunization was followed by full emulsification with the VP7 recombinant protein and subcutaneous injection with equal amount of Freund's incomplete adjuvant at day 14 and day 28. The serum antibody titer of the mice was detected by indirect ELISA, and when the antibody level reached the requirement, the mice were boosted, splenocytes of the mice were aseptically collected 3d after immunization, and then fused with SP2/0 cells.

2. Cell fusion

Prepared SP2/0 cells and mouse spleen cells were prepared according to 1:5, adding 20mL of incomplete culture medium, centrifuging at 1000rpm/min for 10min, and discarding the supernatant. The bottom of the centrifuge tube was gently hit with a finger to disperse the precipitated cells, the cells were fused with 1mL of 50% PEG pre-warmed at 37℃in a 37℃water bath, gently stirred while resting for 1min, terminated with 20mL of DMEM medium, centrifuged at 1000rpm for 5min, the cells were pooled with HAT medium weight suspended melting, inoculated into 96-well cell culture plates, and the 96-well plates were incubated in a 37℃5% CO ₂ incubator.

3. Screening and subcloning of hybridoma cells

HAT medium was used 7 days after fusion, HT medium was used 7-14 days later, and ordinary complete medium was used 14 days later when the area of the clones reached 1/3-1/2 of the area of the culture wells, at which time the medium for all the cloned growth wells was examined. And (5) timely cloning the cells with the detected specific antibody positive holes. Subcloning adopts a double-ratio dilution method, after subcloning for 7 days, detecting culture supernatant by an indirect ELISA method, selecting a single positive hole of a cell mass, continuing subcloning, repeating the process for 3 times, and screening out stable hybridoma cell strains.

The specific method for indirect ELISA detection comprises the following steps:

(1) Coating: AHSV VP7 recombinant protein was diluted to 0.5. Mu.g/mL, 100. Mu.L/well in CBS buffer (0.05M carbonate-bicarbonate buffer, pH 9.6) and coated onto an ELISA plate overnight at 4 ℃. Plates were washed 5 times with PBST buffer.

(2) Closing: the ELISA plates were blocked with 5% nonfat milk in PBST buffer, 200. Mu.L/well, and incubated at 37℃for 1h. Plates were washed 5 times with PBST buffer.

(3) And (3) detection: the hybridoma cell culture supernatant to be detected was added to the ELISA plate at 100. Mu.L/well, while the cell culture supernatant of normal cells was used as a negative control, incubated at 37℃for 1h, and the plate was washed 5 times with PBST buffer.

(4) Adding enzyme-labeled secondary antibodies: 1, 1: a40000 dilution (dilution of 0.01M PBS pH 7.2) of HRP-labeled goat anti-mouse secondary antibody was added to the ELISA plate, 100. Mu.L/well, and incubated at 37℃for 1h. Plates were washed 5 times with PBST buffer.

(5) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; the stop solution was added at 100. Mu.L/well, and the OD ₄₅₀ was read.

The results of OD ₄₅₀ are shown in Table 1, and indicate that the hybridoma cells (designated as 1H4 cell line) have good reactivity with the recombinant VP7 protein.

TABLE 1OD ₄₅₀ detection results

Group of	OD₄₅₀
		1H4 cell line	3.58005
Negative control	0.0757

4. Preparation of mouse ascites antibody

2 Female BALB/c mice of 6-8 weeks old were prepared and liquid paraffin was injected intraperitoneally, 0.5 mL/mouse. After 7 days, 10 ⁶ hybridoma cells obtained in the step 3 are inoculated, the mice are observed, the obvious expansion of the abdomen of the mice is observed about 7-10 days, and when the mice are touched by hands, the skin has tension, so that ascites can be extracted. The collected ascites was centrifuged to remove the precipitate and the supernatant was collected. And then purifying the ascites by using a Protein G column to obtain the 1H4 monoclonal antibody with high titer and high purity. The concentration of the purified monoclonal antibody was 2mg/mL.

The specific purification steps are as follows:

(1) Pretreatment of a chromatographic column: the column volume of 10 times deionized water is washed 3-5 times, and then the column volume of 10 times PBS is washed 3-5 times.

(2) Loading: the ascites was diluted with PBS and filtered through a 022. Mu.L filter.

(3) Washing: PBS was washed until no protein flow was detected (G250 did not change blue).

(4) Antibody elution: elution with 0.1m ph3.0 glycine, the eluate was collected and the eluted product was checked with G250 until it did not turn blue.

(5) PH value adjustment: saturated sodium carbonate adjusts the pH of the eluted product to neutral.

(6) Sample concentration: and (3) a 10kDa ultrafiltration tube, and concentrating the solution to about 1 to 5 mL.

Example 2 identification of monoclonal antibodies against the VP7 protein of African horse sickness Virus

1. Identification of molecular weight and purity of AHSV VP 7-resistant monoclonal antibody

Performing molecular weight and purity identification of the antibody by adopting an SDS-PAGE method; the monoclonal antibody purified in example 1 was prepared, then subjected to SDS-PAGE, loaded with 10. Mu.L per well, stained with Coomassie blue solution for 1h after the electrophoresis was completed, and then decolorized. The results are shown in FIG. 1. The result shows that the monoclonal antibody is successfully purified, the purity is more than 95%, and the application requirement can be met.

2. Determination of the titers of AHSV VP 7-resistant monoclonal antibodies

Determining the titer of the monoclonal antibody by using indirect ELISA, taking AHSV VP7 recombinant protein as a coating antigen, coating at a concentration of 0.5 mug/mL, 100 mug/hole and 4 ℃ overnight; PBST washes the plate 3 times; adding 5% skimmed milk powder for sealing, 200 μl/well, incubating at 37deg.C for 2h, and washing the PBST plate 3 times; respectively adding 100 mu L of a sample to be tested diluted by a ratio (the dilution is 0.01M PBS pH 7.2), incubating for 1h at 37 ℃, and washing the PBST plate for 3 times; adding 1: HRP-labeled goat anti-mouse secondary antibody diluted 10000 (0.01 m PBS ph7.2 as diluent), 100 μl/well, incubated 40min at 37 ℃, pbst washed plates 5 times; the substrate solution (90. Mu.L/well) was added thereto, developed at 37℃in the dark for 10-15min, and the stop solution (50. Mu.L/well) was added thereto. OD ₄₅₀ values were read per well and judged as positive to negative antibody ratio (P/N) greater than 2.1. The results are shown in Table 2, and the titer of the monoclonal antibody can reach 1:1024000, i.e. its dilution concentration is 1.953ng/mL.

TABLE 2 determination of the titers of AHSV VP 7-resistant monoclonal antibodies OD ₄₅₀ values

Concentration of primary antibody	OD₄₅₀
		1ug/mL(1：2000)	4.0883
0.5ug/mL(1：4000)	2.8676
		0.25ug/mL(1：8000)	2.9268
0.125ug/mL(1：16000)	2.709
		62.5ng/mL(1：32000)	2.0886
31.25ng/mL(1：64000)	1.4443
		15.625ng/mL(1：128000)	0.9321
7.813ng/mL(1：256000)	0.5376
		3.906ng/mL(1：512000)	0.2770
1.953ng/mL(1：1024000)	0.1695
		Negative antibodies	0.0378

3. Immunofluorescence detection of AHSV VP 7-resistant monoclonal antibodies

Immunofluorescence analysis was performed on BSR cells transfected with recombinant plasmids expressing AHSV VP7 (pcdna3.1-AHSV VP7, prepared by conventional means) using the prepared anti-AHSV VP7 monoclonal antibodies. The BSR cells were climbing-up, recombinant plasmid pcDNA3.1-AHSV VP7 was transfected into the BSR cells by liposome 3000, incubated at 37℃for 24 hours, cell culture supernatant was discarded, fixed with 4% paraformaldehyde for 10min, washed 3 times with PBS, then subjected to the action of 0.5% Triton X-100 for 10min, washed 3 times with PBS, blocked with 3% BSA for 1 hour, after the old solution was removed by pipetting, anti-AHSV VP7 monoclonal antibody (1:3000) prepared in example 1 was added, incubated overnight at 4℃and then nuclei were stained with Hoechst 33342 for 10min, washed with PBS for 3 times, goat anti-mouse IgG (Alexa was added488 Incubation for 1h at room temperature in the dark, and after sealing, observation and image collection.

As shown in FIG. 2, the BSR cells transfected with the recombinant plasmid pcDNA3.1-AHSV VP7 showed green fluorescence, while the cells transfected with the empty vector pcDNA3.1 did not fluoresce, which indicated that the AHSV VP 7-resistant monoclonal antibodies were able to specifically bind to VP7 protein expressed by eukaryotic cells.

4. Immunoblot detection of AHSV VP 7-resistant monoclonal antibodies

Immunoblotting was performed with a preparation of expressed AHSV VP7 recombinant protein, the primary antibody was incubated with the AHSV VP 7-resistant monoclonal antibody prepared in example 1 (1:3000), washed with PBST 3 times at room temperature for 10 minutes each time, the secondary antibody was anti-mouse with HRP-labeled goat (1:10000), incubated for 1h at room temperature, washed with PBST 3 times, and finally visualized.

As shown in FIG. 3, the AHSV VP 7-resistant monoclonal antibody prepared by the application can specifically react with recombinant VP7 protein in an immunoblotting experiment.

5. Cloning and analysis of monoclonal antibody heavy and light chain variable region genes

(1) Amplification of monoclonal antibody heavy and light chain variable regions

The total RNA of the 1H4 hybridoma cells secreting the AHSV VP 7-resistant monoclonal antibody prepared in example 1 was extracted by TRIzol cleavage method, then cDNA was synthesized by reverse transcription, and then PCR amplification was performed using the cDNA as a template to obtain heavy and light chain variable region nucleotide sequences of the antibody.

Sequencing results show that the nucleotide sequence of the heavy chain variable region of the AHSV VP 7-resistant monoclonal antibody is shown as ：GAGGTGCA GTTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGGTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTTGGTCGCAATAATAAAAAATGATGGTCGTAGTTCCTATTATCCAGACAGTGTGAAGGGCCGATTCACCATCTACAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCACTCTGAAGTCTGAGGACACAGCCATGTATCACTGTGCCTACGGCAGTAGCCTCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO.2);

the amino acid sequence of the heavy chain variable region of the AHSV VP 7-resistant monoclonal antibody is shown as ：EVQLVESGGGLVQPGGSLKLS CAASGFTFSRYGMSWVRQTPDKRLELVAIIKNDGRSSYYPDSVKGRFTIYRDNAKNTLYLQ MSTLKSEDTAMYHCAYGSSLYWYFDVWGAGTTVTVSS(SEQ ID NO.1);

The nucleotide sequence of the light chain variable region of the AHSV VP 7-resistant monoclonal antibody is shown as ：GATGTTTTGATGACCCAAACT CCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGCCAGGTTCAGTGGCAGTGGATCAGGGACAGGTTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAACTGGAAATCAAA(SEQ ID NO.4);

the amino acid sequence of the light chain variable region of the AHSV VP 7-resistant monoclonal antibody is shown as ：DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPARFSGSGSGTGFTLKISRVEAE DLGVYYCFQGSHVPPTFGGGTKLEIK(SEQ ID NO.3).

(2) Determination of CDR regions

Analyzing the sequences of heavy chain and light chain variable regions of the AHSV VP7 resistant monoclonal antibody obtained by sequencing, wherein the amino acid sequences of 3 CDR regions of the heavy chain variable region are respectively as follows:

CDR1: RYGMS (SEQ ID NO. 5);

CDR2: IIKNDGRSSYYPDSVKG (SEQ ID NO. 6);

CDR3: GSSLYWYFDV (SEQ ID NO. 7);

The amino acid sequences of the 3 CDR regions of the light chain variable region are respectively:

CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO. 8); :

CDR2: KVSNSRFS (shown in SEQ ID NO. 9);

CDR3: FQGSHVPPT (SEQ ID NO. 10).

Example 3 establishment of African horse sickness competition ELISA diagnostic method based on an AHSV VP 7-resistant monoclonal antibody

The prepared african horse sickness VP7 recombinant protein was coated overnight at 4℃at a concentration of 0.5. Mu.g/mL and 100. Mu.l/well. African horse sickness standard positive serum and standard negative serum are stored in the laboratory, and the prepared AHSV VP 7-resistant monoclonal antibody is used as a competitive antibody to establish a diagnosis method.

The specific detection method comprises the following steps:

(1) Coating: the expressed AHSV VP7 recombinant protein was diluted to 0.5. Mu.g/mL with CBS buffer (0.05M carbonate-bicarbonate buffer, pH 9.6), 100. Mu.L/well coated into an ELISA plate, coated overnight at 4 ℃; plates were washed 5 times with PBST buffer.

(2) Closing: blocking the ELISA plate with PBST buffer containing 5% skimmed milk powder, 200 μl/well, and incubating at 37deg.C for 1 hr; plates were washed 5 times with PBST buffer.

(3) And (3) detection: african horse sickness positive serum, standard positive and standard negative serum were added to the ELISA plate, 100. Mu.l/well, incubated at 37℃for 1h, and the plate was washed 5 times with PBST buffer.

(4) Then, the AHSV VP 7-resistant monoclonal antibody prepared in example 1 was added at 1:2000 dilution (0.01M PBS pH 7.2), incubated at 37℃for 1h, and the plate was washed 5 times with PBST buffer.

(5) Adding enzyme-labeled secondary antibodies: 1, 1: an HRP-labeled goat anti-mouse secondary antibody diluted with 40000 (0.01M PBS pH 7.2) was added to the ELISA plate, 100. Mu.L/well, and incubated at 37℃for 1h; plates were washed 5 times with PBST buffer.

(6) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; 100. Mu.l/well of stop solution was added and the OD ₄₅₀ was read.

The judging method comprises the following steps: blocking rate (BP) = (negative control OD ₄₅₀ -sample OD ₄₅₀)/(negative control OD ₄₅₀ -positive control OD ₄₅₀) ×100%; the positive control is less than 0.2, the negative control is more than 2.0, and the detection method is established. When BP is less than or equal to 45%, the result is negative; when BP is more than or equal to 50%, the result is positive; when 45% < BP <50%, the results are suspicious.

1. Sensitivity test

African horse sickness positive serum was serially diluted 1:2, 1:4, 1:8, 1:16 to assess the sensitivity of African horse sickness competition ELISA detection methods. The results are shown in Table 3, and the detection result is positive when the serum sample is subjected to 1:16 dilution, which indicates that the African horse sickness competition ELISA detection method established by using the monoclonal antibody 1H4 provided by the application has good sensitivity.

TABLE 3 sensitivity results of African horse sickness competition ELISA detection method

	OD₄₅₀	BP
			1:2	0.2	99.4％
1:4	0.487	88.3％
			1:8	0.881	73.1％
1:16	1.23	59.8％

2. Compliance rate experiment

The results of the competition ELISA test method established in the example for 5 parts (1-5) of AHSV positive serum and 5 parts (6-10) of AHSV negative serum stored in the laboratory are shown in Table 4, and the coincidence rate of the test results of the African horse sickness competition ELISA method established by using the monoclonal antibody 1H4 of the application reaches 100%.

TABLE 4 coincidence rate results of African horse sickness competition ELISA detection method

Sample of	OD₄₅₀	BP
			1	0.5452	84.2％
2	0.3182	93.1％
			3	0.1554	99.5％
4	0.3167	94.9％
			5	1.1153	64.1％
6	2.2916	18.7％
			7	2.2963	18.5％
8	2.7539	0.8％
			9	2.216	21.6％
10	2.6319	5.6％

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A monoclonal antibody that specifically binds to the VP7 protein of the african horse sickness virus, characterized in that the monoclonal antibody comprises an antibody heavy chain and an antibody light chain;

the variable region CDR of the heavy chain of the antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.5, a CDR2 with an amino acid sequence shown as SEQ ID NO.6 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 7;

The variable region CDR of the antibody light chain comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 10.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID No.1 and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID No. 3.

3. A nucleic acid encoding the antibody heavy and light chains of the monoclonal antibody of claim 1 or 2.

4. The nucleic acid of claim 3, wherein the sequence of the gene encoding the variable region of the heavy chain of said antibody is shown in SEQ ID No.2 and the sequence of the gene encoding the variable region of the light chain of said antibody is shown in SEQ ID No. 4.

5. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a reagent, or a test strip, or a kit for detecting african horse sickness virus.

6. An immunoconjugate, the immunoconjugate comprising:

(i) The monoclonal antibody of claim 1 or 2;

7. An african horse sickness virus ELISA detection kit comprising the monoclonal antibody of claim 1 or 2.

8. The ELISA detection kit of claim 7, comprising an ELISA plate, african horse sickness virus antigen, blocking solution, diluent, monoclonal antibody of claim 1 or 2, enzyme-labeled secondary antibody, wash solution, color reagent, stop solution.

9. The ELISA test kit of claim 7, wherein the african horse sickness virus antigen is selected from the group consisting of african horse sickness virus VP7 recombinant proteins.

10. Use of a monoclonal antibody according to claim 1 or 2 for in vitro detection of african horse sickness virus for the purpose of diagnosis of non-disease.